Raffinose saccharides are a group of D-galactose-containing oligosaccharides of sucrose that are widely distributed in plants. Raffinose saccharides are characterized by having the general formula: [0-.beta.-D-galactopyranosyl-(1.fwdarw.6).sub.n -.alpha.-glucopyranosyl-(1.fwdarw.2)-.beta.-D-fructofuranoside where n=0 through n=4 are known respectively as sucrose, raffinose, stachyose, verbascose, and ajugose.
Extensive botanical surveys of the occurrence of raffinose saccharides have been reported in the scientific literature [see Dey, P. M. In Biochemistry of Storage Carbohydrates in Green Plants, Academic Press, London, (1985) pp 53-129]. Raffinose saccharides are thought to be second only to sucrose among the nonstructural carbohydrates with respect to abundance in the plant kingdom. In fact, raffinose saccharides may be ubiquitous, at least among higher plants. Raffinose saccharides accumulate in significant quantities in the edible portion of many economically significant crop species. Examples include soybean (Glycine max L. Merrill), sugar beet (Beta vulgaris), cotton (Gossypium hirsutum L.), canola (Brassica sp.) and all of the major edible leguminous crops including beans (Phaseolus sp.), chick pea (Cicer arietinum), cowpea (Vigna unguiculata), mung bean (Vigna radiata), peas (Pisum sativum), lentil (Lens culinaris) and lupine (Lupinus sp.).
The biosynthesis of raffinose saccharides has been fairly well characterized [see Dey, P. M. In Biochemistry of Storage Carbohydrates in Green Plants (1985)]. The committed reaction of raffinose saccharide biosynthesis involves the synthesis of galactinol (O-.alpha.-D-galactopyranosyl-(1.fwdarw.1)-myo-inositol) from UDP galactose and myo-inositol. The enzyme that catalyzes this reaction is galactinol synthase. Synthesis of raffinose and higher homologues in the raffinose saccliaride family from sucrose is thought to be catalyzed by distinct galactosyltransferases (e.g., raffinose synthase, stachyose synthase, etc.).
Although abundant in many species, raffinose saccharides are an obstacle to the efficient utilization of some economically important crop species. Raffinose saccharides are not digested directly by animals, primarily because .alpha.-galactosidase is not present in the intestinal mucosa [Gitzelmann and Auricchio Pediatrics (1965)36:231-236, Rutloff et al Nahrung. (1967) 11:39-46]. However, microflora in the lower gut are readily able to ferment the raffinose saccharides which results in an acidification of the gut and production of carbon dioxide, methane and hydrogen [Murphy et al. (1972) J. Agr. Food Chem. 20:813-817, Cristofaro et al. In Sugars in Nutrition, (1974) Chapter 20, 313-335, Reddy et al. J. Food Science (1980) 45:1161-1164). The resulting flatulence can severely limit the use of leguminous plants in animal, including human, diets. It is unfortunate that the presence of raffinose saccharides restricts the use of soybeans in animal, including human, diets because otherwise this species is an excellent source of protein and fiber.
The soybean is well-adapted to machinery and facilities for harvesting, storing and processing that are widely available in many parts of the world. In the U.S. alone, approximately 28 million metric tons of meal were produced in 1988 (Oil Crops Situation and Outlook Report, April 1989, U.S. Dept. of Agriculture, Economic Research Service). Typically, hulls are removed and then the oil is extracted with hexane in one of several extraction systems. The remaining defatted flakes can then be used for a variety of commercial soy protein products [see Soy Protein Products, Characteristics, Nutritional Aspects and Utilization (1987) Soy Protein Council]. Foremost among these in volume of use is soybean meal, the principle source of protein in diets used for animal feed, especially those for monogastric animals such as poultry and swine.
Although the soybean is an excellent source of vegetable protein, there are inefficiencies associated with its use that appear to be due to the presence of raffinose saccharides. Compared to maize, the other primary ingredient in animal diets, gross energy utilization for soybean meal is low [see Potter and Potchanakorn In Proceedings World Soybean Conference III, (1984) 218-224]. For example, although soybean meal contains approximately 6% more gross energy than ground yellow corn, it has about 40 to 50% less metabolizable energy when fed to chickens. This inefficiency of gross energy utilization does not appear to be due to problems in digestion of the protein fraction of the meal, but rather due to the poor digestion of the carbohydrate portion of the meal. It has been reported that removal of raffinose saccharides from soybean meal by ethanol extraction results in a large increase in the metabolizable energy for broilers [Coon, C. N. et al. Proceedings Soybean Utilization Alternatives, University of Minnesota, (1988) 203-211]. Removal of the raffinose saccharides was associated with increased utilization of the cellulosic and hemicellulosic fractions of the soybean meal.
A variety of processed vegetable protein products are produced from soybean. These range from minimally processed, defatted items such as soybean meal, grits, and flours to more highly processed items such as soy protein concentrates and soy protein isolates. In other soy protein products the oil is not extracted, full-fat soy flour for example. In addition to these processed products, there are also a number of speciality products based on traditional Oriental processes, which utilize the entire bean as the starting material. Examples include soy milk, soy sauce, tofu, natto, miso, tempeh, and yuba.
Examples of use of soy protein products in human foods include soy protein concentrates, soy protein isolates, textured soy protein, soy milk, and infant formula. Facilities and methods to produce protein concentrates and isolates from soybeans are available across the world. One of the problems faced by producers of soy protein concentrates and isolates is the challenge of selectively purifying the protein away from the raffinose saccharides. Considerable equipment and operating costs are incurred as a result of removing the large amounts of raffinose saccharides that are present in soybeans.
The problems and costs associated with raffinose saccharides could be reduced or eliminated through the availability of genes that confer a reduction of raffinose saccharide content of soybean seeds. Such genes could be used to develop soybean varieties having inherently reduced raffinose saccharide content. Soybean varieties with inherently reduced raffinose saccharide content would improve the nutritional quality of derived soy protein products and reduce processing costs associated with the removal of raffinose saccharides. Said low raffinose saccharide soybean varieties would be more valuable than conventional varieties for animal and human diets and would allow mankind to more fully utilize the desirable nutritional qualities of this edible legume.
Efforts have been made to identify soybean germplasm that may contain genes that confer a low seed raffinose saccharide content phenotype. Surveys of the soybean germplasm collection, including Glycine max, Glycine soja, and Glycine hirsutum, tentatively identified PI lines that seemed to offer the potential for reducing raffinose saccharide content via conventional breeding [see Hymowitz, T., et al. Comm. In Soil Science and Plant Analysis (1972) 3:367-373, Hymowitz, T., et al. Agronomy J. (1972) 64:613-616, Hymowitz, T., and Collins, F. I. Agronomy J. (1974) 66:239-240, Openshaw, S. J., and Hadley, H. H. Crop Science (1978) 18:581-584, Openshaw, S. J., and Hadley, H. H. Crop Science (1981) 21:805-808, and Saravitz (1986) Ph.D. Thesis, North Carolina State University, Horticultural Science Department]. However, when assayed under identical analytical conditions, none of the lines suggested in these prior surveys proved to be significantly lower in raffinose saccharide content than the currently available elite soybean lines. The primary reason for this may be due to the instability of the low raffinose saccharide phenotype. Results from germplasm collection surveys are highly influenced by the quality of the seed obtained from the collection. This is particularly true for raffinose saccharides in that seed carbohydrate composition has been shown to be influenced by seasonal, genetic and environmental factors [Jacorzynski, B. and Barylko-Pikielna, N. Acta Agrobotanica (1983)36:41-48, Saravitz (1986) Ph.D. Thesis, North Carolina State University, Horticultural Science Department]. Furthermore, seed storage conditions prior to analysis can also influence the composition [Ovacharov and Koshelev Fiziol. Rast. (1974) 21:969-974, Caffrey et al. Plant Physiol. (1988) 86:754-758, Schleppi and Burns Iowa Seed Science (1989) 11:9-12]. As a result, the potential exists for falsely identifying soybean germplasm whose reduced raffinose saccharide content is not heritable, but rather due to the environment in which the seeds were produced or stored prior to analysis. Collectively, these factors have severely limited efforts to identify soybean genes that reduce raffinose saccharide content.
The difficulty and unreliability of screens for raffinose saccharide content is reflected by the paucity of publicly available soybean carbohydrate data as compared to protein and oil quality data. For example, the USDA has numerous publications revealing the protein and oil quality contents for almost all (ca. 14,000) of the soybean PI lines in the USDA collection. However, although raffinose saccharide content is known to be a serious problem in soybeans, very little of the PI collection has actually been screened for this trait.
Demonstration of the stability of a low raffinose saccharide phenotype in subsequent generations (heritability of the phenotype) is required if the germplasm is to be of any utility in improving seed quality. It is therefore essential that any putative germplasm source be regrown to obtain fresh seed and reassayed (with appropriate lines as experimental controls) before it is declared as a potential source of low raffinose sacclaride genes. Once the heritability (stability) of the phenotype is demonstrated, it is desirable to determine the inheritance (number and nature of genes that are involved) of the low raffinose saccharide phenotype. Heritability and inheritance information is extremely valuable for attempts to breed new soybean varieties that contain the low raffinose saccharide trait.
In light of the above described factors, it is apparent that soybean plants with heritable, substantially reduced raffinose saccharide content useful for preparing soy protein products with an improved carbohydrate content are needed. Heretofore, the only means to acheive a desirable raffinose saccharide content was to physically and/or chemically treat the soybean.